SIST EN 1276:2010
(Main)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
This document specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or - in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance.
This document applies to products that are used in food, industrial, domestic and institutional areas excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas. The following areas are at least included:
a) processing, distribution and retailing of:
1) food of animal origin:
milk and milk products;
meat and meat products;
fish, seafood, and related products;
eggs and egg products;
animal feeds;
etc. 2) food of vegetable origin:
beverages;
fruits, vegetables and derivatives (including sugar, distillery, etc.);
flour, milling and baking;
animal feeds;
etc.
b) institutional and domestic areas:
catering establishments;
public areas;
public transports;
schools;
nurseries;
shops;
sports rooms;
waste containers (bins, etc.);
hotels;
dwellings;
clinically non sensitive areas of hospitals;
offices;
etc.
c) other industrial areas:
packaging material;
biotechnology (yeast, proteins, enzymes, etc.);
pharmaceutical;
cosmetics and toiletries;
textiles;
space industry, computer industry;
etc.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Diese Europäische Norm legt ein Prüfverfahren für und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsprodukten und antiseptischen Produkten fest, die bei Verdünnung in Wasser standardisierter Härte oder — im Falle gebrauchsfertiger Produkte — in Wasser als homogenes und physikalisch stabiles Präparat vorliegen. Die Produkte können nur bei einer Konzentration von höchstens 80 % geprüft werden, da durch die Zugabe der Prüfkeime und der Belastungssubstanz immer eine bestimmte Verdünnung auftritt.
Das vorliegende Dokument gilt für Produkte, die im Lebensmittelbereich, in der Industrie, im Haushalt und in öffentlichen Einrichtungen genutzt werden; ausgenommen sind Bereiche und Situationen, in denen die Desinfektion medizinisch angezeigt ist und Produkte, die auf lebenden Geweben verwendet werden, hier wiederum außer denen, die zur Handhygiene in den vorstehend genannten Bereichen verwendet werden. Es werden mindestens folgende Bereiche abgedeckt:
a) Verarbeitung, Vertrieb und Verkauf von:
1) Lebensmitteln tierischer Herkunft:
- Milch und Milcherzeugnisse;
- Fleisch und Fleischerzeugnisse;
- Fisch, Meerestiere und zugehörige Erzeugnisse;
- Eier und Eierzeugnisse;
- Tiernahrung;
- usw.; 2) Lebensmitteln pflanzlicher Herkunft:
- Getränke;
- Obst, Gemüse und zugehörige Erzeugnisse (einschließlich Zucker, Destillationsprodukte usw.);
- Mehl, gemahlene und gebackene Erzeugnisse;
- Tiernahrung;
- usw.;
b) öffentliche Einrichtungen und Haushalt:
- Verpflegungsbetriebe;
- öffentliche Bereiche;
- öffentliche Verkehrsmittel;
- Schulen;
- Kindergärten, Kinderkrippen u. Ä.;
- Geschäfte;
- Sportstätten;
- Abfallbehälter (Mülltonnen usw.);
- Hotels;
- Wohngebäude;
- klinisch nicht kritische Bereiche in Krankenhäusern;
- Büroräume;
- usw.;
c) weitere industrielle Bereiche:
- Verpackungsmaterial;
- Biotechnologie (Hefe, Proteine, Enzyme, usw.);
- Pharmazeutika;
- Kosmetika und Toilettenartikel;
- Textilien;
- Luft und R
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)
La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l'eau dure ou - dans le cas de produits prêts à l’emploi - dans l’eau. Les produits ne peuvent être soumis à l’essai qu’à la concentration de 80 % ou à des concentrations inférieures, car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente norme s'applique aux produits utilisés dans les domaines alimentaire, industriel, domestique et en collectivité à l'exclusion des domaines et des situations où la désinfection est médicalement prescrite et à l'exclusion des produits utilisés sur les tissus vivants (excepté ceux destinés à l'hygiène des mains dans les domaines indiqués ci-dessus). La présente Norme européenne s'applique au moins aux domaines suivants :
a) La transformation, la distribution et le commerce de détail des :
1) aliments d'origine animale :
- lait et produits laitiers ;
- viande et produits carnés ;
- poisson, fruits de mer et leurs dérivés ;
- œufs et produits dérivés ;
- alimentation animale ;
- etc. 2) aliments d'origine végétale :
- boissons ;
- fruits, légumes et leurs dérivés (y compris le sucre, et les produits de distillation, etc.) ;
- farine, minoterie et boulangerie ;
- alimentation animale ;
- etc.
b) les domaines domestique et la collectivité :
- établissements de restauration ;
- lieux publics ;
- transports publics ;
- écoles ;
- crèches ;
- magasins ;
- salles de sport ;
- conteneurs pour déchets (poubelles, etc.) ;
- hôtels ;
- locaux d'habitation ;
- zones cliniquement non sensibles des hôpitaux ;
- bureaux ;
- etc.
c) Autres domaines industriels :
- les matériaux d'emballage ;
- la biotechnologie (levure, protéines, enzymes, etc.) ;
- l'industrie pharmaceutique ;
- (...)
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja 1)
Ta evropski standard opredeljuje preskusno metodo in minimalne zahteve baktericidnega delovanja kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizično stabilen pripravek, razredčen s trdo vodo ali – v primeru končnega proizvoda – z vodo. Proizvod je lahko preskušen samo pri koncentraciji 80 % ali manj, ker je določeno redčenje vedno doseženo s tem, ko dodajamo testni organizem in motečo snov. Ta dokument velja za produkte, ki so v uporabi v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah, pri čemer so izvzeta področja in okoliščine, kjer je dezinfekcija zdravstveno pogojena, ter izvzeti proizvodi, ki se uporabljajo za živa tkiva, razen tistih, ki se uporabljajo za higieno rok na zgoraj navedenih področjih. Vsaj naslednja področja so vključena: a) predelava, distribucija in maloprodaja: 1) hrana živalskega izvora: mleko in mlečni proizvodi; meso in mesni proizvodi; ribe, morska hrana in s tem povezani proizvodi; jajca in jajčni proizvodi; živalska krmila itd. 2) hrana rastlinskega izvora: pijače; sadja, zelenjava in njihovi proizvodi (vključujoč sladkor, žganja itd.); moka, mletje in peka; živalska krmila itd.; b) področja javnih ustanov in gospodinjstev; ustanovitev cateringa; javna območja; javni prevoz; šole; drevesnice; trgovine; športni objekti; zabojniki za odpadke (posode, itd.); hoteli; stanovanja; klinično neobčutljiva področja v bolnicah; pisarne itd. EN 1276:2009 (E) 7; c) ostala industrijska področja: material za embalažo; biotehnologija (kvas, proteini, encimi itd.); farmacevtika, kozmetika in toaletni izdelki; tekstili; vesoljska industrija; računalniška industrija itd. EN 14885 podrobno opredeljuje razmerje med različnimi preskusi in »priporočili uporabe«.
General Information
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposesICS:Ta slovenski standard je istoveten z:EN 1276:2009SIST EN 1276:2010en,fr,de01-januar-2010SIST EN 1276:2010SLOVENSKI
STANDARDSIST EN 1276:20011DGRPHãþD
SIST EN 1276:2010
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1276
October 2009 ICS 71.100.35 Supersedes EN 1276:1997English Version
Chemical disinfectants and antiseptics -Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (Phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 20 September 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 1276:2009: ESIST EN 1276:2010
EN 1276:2009 (E) 2 Contents Page Foreword .4Introduction .51Scope .62Normative references .73Terms and definitions .74Requirements .75Test method .85.1Principle .85.2Materials and reagents .95.2.1Test organisms .95.2.2Culture media and reagents .95.3Apparatus and glassware . 135.4Preparation of test organism suspensions and product test solutions . 145.4.1Test organism suspensions (test and validation suspension) . 145.4.2Product test solutions . 155.5Procedure for assessing the bactericidal activity of the product . 175.5.1General . 175.5.2Dilution-neutralization method . 185.5.3Membrane filtration method . 205.6Experimental data and calculation. 225.6.1Explanation of terms and abbreviations . 225.6.2Calculation . 235.7Verification of methodology . 265.7.1General . 265.7.2Control of weighted mean counts . 265.7.3Basic limits . 265.8Expression of results and precision . 265.8.1Reduction . 265.8.2Control of active and non-active product test solution (5.4.2) . 275.8.3Limiting test organism and bactericidal concentration . 275.8.4Precision, replicates . 275.9Interpretation of results - conclusion . 275.9.1General . 275.9.2Bactericidal activity for general purposes . 27SIST EN 1276:2010
EN 1276:2009 (E) 3 5.9.3Bactericidal activity for specific purposes . 275.10Test report . 28Annex A (informative)
Referenced strains in national collections . 30Annex B (informative)
Neutralizers and rinsing liquids . 31Annex C (informative)
Graphical representations of dilution neutralization method and membrane filtration method . 33C.1Dilution-neutralization method . 33C.2Membrane filtration method . 35Annex D (informative)
Example of a typical test report . 37Test results (bactericidal suspension test) . 39Annex E (informative)
Precision of the test result . 41Bibliography . 44 SIST EN 1276:2010
EN 1276:2009 (E) 4 Foreword This document (EN 1276:2009) has been prepared by Technical Committee CEN/TC 216 “Chemical disinfectants and antiseptics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 2010, and conflicting national standards shall be withdrawn at the latest by April 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 1276:1997. It was revised to include the results of a collaborative trial (ANDISTAND), to correct obvious errors and ambiguities, to harmonize the structure and wording with other quantitative suspension tests of CEN/TC 216 (existing or in preparation) and to improve the readability of the standard and thereby make it more understandable. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 1276:2010
EN 1276:2009 (E) 5 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have bactericidal activity in the fields described in the scope. This laboratory test takes into account practical conditions of application of the product, including contact time, temperature, test organisms and interfering substance, i.e. conditions which may influence its action in practical situations. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test corresponds to defined experimental conditions. However, for some applications, the recommendations of use of a product may differ and therefore additional test conditions need to be used. SIST EN 1276:2010
EN 1276:2009 (E) 6 1 Scope This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or - in the case of ready-to-use products - with water. Products can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance. This document applies to products that are used in food, industrial, domestic and institutional areas excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas. The following areas are at least included: a) processing, distribution and retailing of: 1) food of animal origin: milk and milk products; meat and meat products; fish, seafood, and related products; eggs and egg products; animal feeds; etc. 2) food of vegetable origin: beverages; fruits, vegetables and derivatives (including sugar, distillery, etc.); flour, milling and baking; animal feeds; etc. b) institutional and domestic areas: catering establishments; public areas; public transports; schools; nurseries; shops; sports rooms; waste containers (bins, etc.); hotels; dwellings; clinically non sensitive areas of hospitals; offices; etc. SIST EN 1276:2010
EN 1276:2009 (E) 7 c) other industrial areas: packaging material; biotechnology (yeast, proteins, enzymes, etc.); pharmaceutical; cosmetics and toiletries; textiles; space industry, computer industry; etc. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity EN 14885:2006, Chemical disinfectants and antiseptics — Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885:2006 apply. 4 Requirements The product shall demonstrate at least a 5 decimal log (lg) reduction when diluted with hard water (5.2.2.7) or - in the case of ready-to-use products - with water (5.2.2.2) and tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution- 5.2.2.8.2) or simulated dirty conditions (3 g/l bovine albumin solution - 5.2.2.8.3) according to its practical applications and under the other obligatory test conditions (four selected test organisms, 20 °C, 5 min or 1 min (for hands disinfection)). The bactericidal activity shall be evaluated using the following four test organisms: Pseudomonas aeruginosa; Escherichia coli; SIST EN 1276:2010
EN 1276:2009 (E) 8 Staphylococcus aureus; Enterococcus hirae. Where indicated, additional specific bactericidal activity shall be determined applying other contact times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into account intended specific use conditions. NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. 5 Test method 5.1 Principle 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is maintained at (20 ± 1) °C for 5 min ± 10 s (obligatory test conditions) during 1 min (obligatory test conditions for hands disinfection). At the end of this contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in each sample are determined and the reduction is calculated. 5.1.2 The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae as test organisms. 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be used. SIST EN 1276:2010
EN 1276:2009 (E) 9 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms1): Pseudomonas aeruginosa ATCC 15442;
Escherichia coli ATCC 10536;
Staphylococcus aureus ATCC 6538;
Enterococcus hirae ATCC 10541. If required for specific applications, additional strains may be chosen, for example from: Salmonella Typhimurium ATCC 13311;
Lactobacillus brevis DSM 6235;
Enterobacter cloacae DSM 6234. NOTE See Annex A for strain references in some other culture collections. The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years. 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed. NOTE 2 For each culture medium and reagent, a limitation for use should be fixed.
1)
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC). This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named. SIST EN 1276:2010
EN 1276:2009 (E) 10 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralized water. Sterilize in the autoclave (see 5.3.2.1 a). NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized. NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. NOTE 3 See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein
15,0 g Soya peptone, papaic digest of soybean meal
5,0 g Sodium chloride (NaCl)
5,0 g Agar
15,0 g Water (5.2.2.2)
to 1 000,0 ml Sterilize in the autoclave (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. NOTE In the case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of: Tryptone, pancreatic digest of casein
1,0 g Sodium chloride (NaCl)
8,5 g Water (5.2.2.2)
to 1 000,0 ml Sterilize in the autoclave (5.3.2.1 a). After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C. 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B. SIST EN 1276:2010
EN 1276:2009 (E) 11 5.2.2.6 Rinsing liquid (for membrane filtration) The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.7 Hard water for dilution of products For the preparation of 1 000 ml of hard water, the procedure is as follows: prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
(5.3.2.1 a). Autoclaving – if used - may cause a loss of liquid. In this case, make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one month; prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no longer than one week; place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.4). If necessary, adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl). The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces a different final water hardness in each test tube. In any case, the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. The interfering substance shall be sterile and prepared at 10 times its final concentration in the test. The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l. SIST EN 1276:2010
EN 1276:2009 (E) 12 5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration) Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l. 5.2.2.8.4 Milk (dairies, etc.) Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per litre of water (5.2.2.2), shall be prepared as follows: prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2). Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3) °C]. The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk. 5.2.2.8.5 Yeast extract (breweries, etc.) Dehydrated yeast extract for bacteriology, shall be prepared as follows: prepare a 100 g/l solution in water (5.2.2.2), adjust to pH 7,0 ± 0,2 with sodium hydroxide (NaOH); sterilize in the autoclave (5.3.2.1 a). The final concentration of yeast extract in the test procedure (5.5) is 10,0 g/l. 5.2.2.8.6 Sucrose (beverage, soft drink industries) Prepare a 100 g/l solution of sucrose in water (5.2.2.2), sterilize by membrane filtration (5.3.2.7). The final concentration of sucrose in the test procedure (5.5) is 10,0 g/l. 5.2.2.8.7 pH 5,0 and pH 9,0 buffer solutions (cleaning in place, etc.) The buffer solution used shall be described in the test report and pH values shall be recorded. The final pH in the test tubes (together with test organisms and product) shall be controlled and found equal to 5,0 ± 0,2 or 9,0 ± 0,2. 5.2.2.8.8 Sodium dodecyl sulphate (cosmetic area, etc.) Prepare a 50 g/l solution of sodium dodecyl sulphate (C12H25NaO4S) in water (5.2.2.2). Sterilize in the autoclave (5.3.2.1 a). The final concentration of sodium dodecyl sulphate in the test procedure (5.5) is 5,0 g/l. SIST EN 1276:2010
EN 1276:2009 (E) 13 5.3 Apparatus and glassware 5.3.1 General Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: a) by moist heat, in the autoclave (5.3.2.1 a); b) by dry heat, in the hot air oven (5.3.2.1 b). 5.3.2 Usual microbiological laboratory equipment 2) and, in particular, the following: 5.3.2.1 Apparatus for sterilization: a) for moist heat sterilization, an autoclave capable of being maintained at C )121(30°+ for a minimum holding time of 15 min; b) for dry heat sterilization, a hot air oven capable of being maintained at C )180(50°+ for a minimum holding time of 30 min, at C )170(50°+ for a minimum holding time of 1 h or at C )160(50°+ for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted TSA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled either at (36 ± 1) °C or (37 ± 1) °C (5.2.1). 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch 5.3.2.6 Shaker a) Electromechanical agitator, e.g. Vortex® mixer3). b) Mechanical shaker 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin (5.2.2.8.2 and 5.2.2.8.3) and sucrose (5.2.2.8.6), and if the membrane filtration method is used (5.5.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 1276:2010
EN 1276:2009 (E) 14 5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated automatic pipettes. 5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm. 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 General For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the test a
...
SLOVENSKI STANDARD
kSIST FprEN 1276:2009
01-april-2009
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Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial,
domestic and institutional areas - Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in
den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques
utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines
domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: FprEN 1276
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
kSIST FprEN 1276:2009 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST FprEN 1276:2009
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kSIST FprEN 1276:2009
EUROPEAN STANDARD
FINAL DRAFT
FprEN 1276
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2009
ICS 11.080 Will supersede EN 1276:1997
English Version
Chemical disinfectants and antiseptics - Quantitative suspension
test for the evaluation of bactericidal activity of chemical
disinfectants and antiseptics used in food, industrial, domestic
and institutional areas - Test method and requirements (phase
2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif Chemische Desinfektionsmittel und Antiseptika -
de suspension pour l'évaluation de l'activité bactéricide des Quantitativer Suspensionsversuch zur Bestimmung der
antiseptiques et des désinfectants chimiques utilisés dans bakteriziden Wirkung chemischer Desinfektionsmittel und
le domaine de l'agro-alimentaire, dans l'industrie, dans les Antiseptika in den Bereichen Lebensmittel, Industrie,
domaines domestiques et en collectivité - Méthode d'essai Haushalt und öffentliche Einrichtungen - Prüfverfahren und
et prescriptions (phase 2, étape 1) Anforderungen (Phase 2, Stufe 1)
This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical
Committee CEN/TC 216.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which
stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other language
made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the
same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprEN 1276:2009: E
worldwide for CEN national Members.
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FprEN 1276:2009 (E)
Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .6
3 Terms and definitions .6
4 Requirements .7
5 Test method .7
Annex A (informative) Referenced strains in national collections . 29
Annex B (informative) Suitable neutralizers and rinsing liquids . 30
Annex C (informative). 32
Annex D (informative) Example of a typical test report . 36
Annex E (informative) Precision of the test result . 40
Bibliography . 43
2
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FprEN 1276:2009 (E)
Foreword
This document (FprEN 1276:2009) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the Unique Acceptance Procedure.
This document will supersede EN 1276:1997.
It was revised to include the results of a collaborative trial (ANDISTAND), to correct obvious errors and
ambiguities, to harmonize the structure and wording with other quantitative suspension tests of CEN/TC 216
(existing or in preparation) and to improve the readability of the standard and thereby make it more
understandable.
3
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FprEN 1276:2009 (E)
Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or
antiseptic has or does not have bactericidal activity in the fields described in the scope.
This laboratory test takes into account practical conditions of application of the product, including contact time,
temperature, test organisms and interfering substance, i.e. conditions which may influence its action in
practical situations.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined experimental conditions. However, for some applications, the recommendations of use
of a product may differ and therefore additional test conditions need to be used.
4
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FprEN 1276:2009 (E)
1 Scope
This document specifies a test method and the minimum requirements for bactericidal activity of chemical
disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with
hard water or - in the case of ready-to-use products - with water. Products can only be tested at a
concentration of 80 % or less, as some dilution is always produced by adding the test organisms and
interfering substance.
This document applies to products that are used in food, industrial, domestic and institutional areas excluding
areas and situations where disinfection is medically indicated and excluding products used on living tissues
except those for hand hygiene in the above considered areas. The following areas are at least included:
a) processing, distribution and retailing of:
1) food of animal origin: 2) food of vegetable origin:
milk and milk products; beverages;
meat and meat products; fruits, vegetables and derivatives
(including sugar, distillery, etc.);
fish, seafood, and related products;
flour, milling and baking;
eggs and egg products;
animal feeds;
animal feeds;
etc.
etc.
b) institutional and domestic areas:
catering establishments;
public areas;
public transports;
schools;
nurseries;
shops;
sports rooms;
waste containers (bins, etc.);
hotels;
dwellings;
clinically non sensitive areas of hospitals;
offices;
etc.
5
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kSIST FprEN 1276:2009
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c) other industrial areas:
packaging material;
biotechnology (yeast, proteins, enzymes, etc.);
pharmaceutical;
cosmetics and toiletries;
textiles;
space industry, computer industry;
etc.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances
under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the
determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity
EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical
disinfectants and antiseptics
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
product
chemical agent or formulation used as a chemical disinfectant or antiseptic
3.2
bactericide
product that kills vegetative bacteria under defined conditions
NOTE The adjective derived from “bactericide” is “bactericidal”.
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3.3
bactericidal activity
capability of a product to produce a reduction in the number of viable bacterial cells of relevant test organisms
under defined conditions
3.4
bacteriostatic activity
capability of a product to inhibit the growth of bacteria under defined conditions
4 Requirements
The product shall demonstrate at least a 5 decimal log (lg) reduction when diluted with hard water (5.2.2.7)
or - in the case of ready-to-use products - with water (5.2.2.2) and tested in accordance with Clause 5 under
simulated clean conditions (0,3 g/l bovine albumin solution- 5.2.2.8.2) or simulated dirty conditions (3 g/l
bovine albumin solution - 5.2.2.8.3) according to its practical applications and under the other obligatory test
conditions (four selected test organisms, 20 °C, 5 min).
The bactericidal activity shall be evaluated using the following four test organisms:
Pseudomonas aeruginosa;
Escherichia coli;
Staphylococcus aureus;
Enterococcus hirae.
Where indicated, additional specific bactericidal activity shall be determined applying other contact times,
temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in
order to take into account intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained
under the obligatory test conditions.
5 Test method
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use
products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is
maintained at (20 ± 1) °C for 5 min ± 10 s (obligatory test conditions). At the end of this contact time, an
aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized
or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer
cannot be found, membrane filtration is used. The numbers of surviving bacteria in each sample are
determined and the reduction is calculated.
5.1.2 The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and
Enterococcus hirae as test organisms.
5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can
be used.
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5.2 Materials and reagents
5.2.1 Test organisms
)
1
The bactericidal activity shall be evaluated using the following strains as test organisms :
Pseudomonas aeruginosa ATCC 15442;
ATCC 10536;
Escherichia coli
ATCC 6538;
Staphylococcus aureus
ATCC 10541.
Enterococcus hirae
If required for specific applications, additional strains may be chosen, for example from:
ATCC 13311;
Salmonella Typhimurium
DSM 6235;
Lactobacillus brevis
DSM 6234.
Enterobacter cloacae
NOTE See Annex A for strain references in some other culture collections.
The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The same
temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control
and validation.
If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature,
time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond
to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional
test organisms are not classified at a reference centre, their identification characteristics shall be stated. In
addition, they shall be held by the testing laboratory or national culture collection under a reference for five
years.
5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated
forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free
from substances that are toxic or inhibitory to the test organisms.
NOTE 1 To improve reproducibility, it is recommended that commercially available dehydrated material is used for the
preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be
rigorously followed.
NOTE 2 For each culture medium and reagent, a limitation for use should be fixed.
)
1
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC).
This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of
the product named.
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5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave (see 5.3.2.1 a).
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can
be used.
NOTE 3 See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Tryptone Soya Agar (TSA)
Tryptone soya agar, consisting of:
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to
7,2 ± 0,2 when measured at (20 ± 1) °C.
NOTE In the case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add
neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used.
5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein
1,0 g
Sodium chloride (NaCl)
8,5 g
Water (5.2.2.2)
to 1 000,0 ml
Sterilize in the autoclave (5.3.2.1 a). After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2
when measured at (20 ± 1) °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It
shall be sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in
Annex B.
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5.2.2.6 Rinsing liquid (for membrane filtration)
The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.3.
It shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane
under the test conditions described in 5.5.3.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in
Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 000 ml of hard water, the procedure is as follows:
prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride (CaCl ) in
2 2
water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
(5.3.2.1 a). Autoclaving – if used - may cause a loss of liquid. In this case, make up to 1 000 ml with water
(5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one
month;
prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
3
1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no
longer than one week;
place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9)
of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH of the hard
water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces a
different final water hardness in each test tube. In any case, the final hardness is lower than 300 mg/l of calcium carbonate
(CaCO ) in the test tube.
3
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral
substances, protein, carbohydrates, lipids and detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l.
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5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.
5.2.2.8.4 Milk (dairies, etc.)
Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder per
litre of water (5.2.2.2), shall be prepared as follows:
prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2). Heat for 30 min at (105 ± 3) °C [or
5 min at (121 ± 3) °C].
The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk.
5.2.2.8.5 Yeast extract (breweries, etc.)
Dehydrated yeast extract for bacteriology, shall be prepared as follows:
prepare a 100 g/l solution in water (5.2.2.2), adjust to pH 7,0 ± 0,2 with sodium hydroxide (NaOH);
sterilize in the autoclave (5.3.2.1 a).
The final concentration of yeast extract in the test procedure (5.5) is 10,0 g/l.
5.2.2.8.6 Sucrose (beverage, soft drink industries)
Prepare a 100 g/l solution of sucrose in water (5.2.2.2), sterilize by membrane filtration (5.3.2.7).
The final concentration of sucrose in the test procedure (5.5) is 10,0 g/l.
5.2.2.8.7 pH 5,0 and pH 9,0 buffer solutions (cleaning in place, etc.)
The buffer solution used shall be described in the test report and pH values shall be recorded. The final pH in
the test tubes (together with test organisms and product) shall be controlled and found equal to 5,0 ± 0,2 or
9,0 ± 0,2.
5.2.2.8.8 Sodium dodecyl sulphate (cosmetic area, etc.)
Prepare a 50 g/l solution of sodium dodecyl sulphate (C H NaO S) in water (5.2.2.2). Sterilize in the
12 25 4
autoclave (5.3.2.1 a).
The final concentration of sodium dodecyl sulphate in the test procedure (5.5) is 5,0 g/l.
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FprEN 1276:2009 (E)
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave (5.3.2.1 a);
b) by dry heat, in the hot air oven (5.3.2.1 b).
2)
5.3.2 Usual microbiological laboratory equipment and, in particular, the following:
5.3.2.1 Apparatus for sterilization:
+3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum
0
holding time of 15 min;
+5
(180 ) °C for a minimum holding
b) for dry heat sterilization, a hot air oven capable of being maintained at
0
+5 +5
time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a minimum holding
0 0
time of 2 h.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted TSA in
case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).
5.3.2.3 Incubator, capable of being controlled either at (36 ± 1) °C or (37 ± 1) °C (5.2.1).
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
NOTE A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media
(5.2.2.3).
5.3.2.5 Stopwatch
5.3.2.6 Shaker
® 3)
a) Electromechanical agitator, e.g. Vortex mixer .
b) Mechanical shaker
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be
filtered.
The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of
diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin
(5.2.2.8.2 and 5.2.2.8.3) and sucrose (5.2.2.8.6), and if the membrane filtration method is used (5.5.3).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the
micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain
the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
2)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
3) ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of
users of this standard and does not constitute an endorsement by CEN of this product.
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FprEN 1276:2009 (E)
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated automatic
pipettes.
5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
5.3.2.11 Glass beads, 3 mm to 4 mm in diameter.
5.3.2.12 Volumetric flasks
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
5.4.1.1 General
For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the
test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
5.4.1.3 Working culture of test organisms
In order to prepare the working culture of the test organisms (5.2.1), prepare a subculture from the stock
culture (5.4.1.2) by streaking onto TSA slopes (5.2.2.3) or plates (5.3.2.10) and incubate (5.3.2.3). After 18 h
to 24 h prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h.
From this second subculture, a third subculture may be produced in the same way. The second and (if
produced) third subcultures are the working cultures.
If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used for
subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 48 h
period.
Never produce and use a fourth subculture.
For additional test organisms, any departure from this method of culturing the test organisms or preparing the
suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (“N”)
a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the
working culture (5.4.1.3) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be
suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge the cells before
immersing in the diluent. Shake the flask for 3 min using a mechanical shaker (5.3.2.6 b). Aspirate the
suspension from the glass beads and transfer to another tube.
8 8
4)
Adjust the number of cells in the suspension to (1,5 x 10 ) cfu/ml to (5 x 10 )cfu/ml using diluent
(5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water
bath at the test temperature θ (5.5.1.1 a) and use within 2 h.
4)
cfu/ml = colony forming unit(s) per millilitre.
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kSIST FprEN 1276:2009
FprEN 1276:2009 (E)
NOTE The use of spectrophotometer for adjusting the number of cells is highly recommended (approximately 620 nm
wavelength - cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each test
organism knowing that suitable values of optical density are generally found beween 0,150 and 0,460. A colorimeter is a
suitable alternative.
-6 -7
b) For counting, prepare 10 and 10 dilutions of the test suspension using diluent (5.2.2.4). Mix (5.3.2.6 a).
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread
plate technique.
1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml melted TSA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size – on an appropriate number (at least two) of surface dried plates containing
TSA (5.2.2.3).
For incubation and count
...
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